The safety and efficacy of protein biologics has led to an upswing in the number of such drugs being introduced into development pipelines toward clinical use. Additional benefits of protein drugs will be realized by way of their application for specialized needs that include both precision and personalized medicine, treatment of orphan diseases, and point-of-care delivery of medical products. Currently, the manufacture of protein biologics is primarily implemented with large scale heterologous expression in bacterial, yeast, and mammalian cell-based systems. Problematically, cell-based gene expression and protein folding are host-dependent, and optimization for product export into the culture fluid is often required to improve production. Other limitations of protein production in living cells include: formation of insoluble protein aggregates, protein degradation by intracellular proteases, and in some cases, inadequate target expression due to host cell toxicity of over-expressed protein and/or an inability to confer appropriate humanized post-translational modifications (PTMs) in target proteins. Overall, such cell-based approaches are impractical for cost-effective and rapid manufacture of low doses of protein biologics and/or production at the point of need because they require multiple processes using large bioreactors, specialized facilities, lengthy production cycles, optimum and stable conditions for sustainable cell growth, laborious purification protocols, all resulting in long turnaround times between cell transfection and protein isolation as well as high costs (Spirin 2004, Trends Biotechnol. 22:538-545; Gilbert and Albala, 2002, Curr. Opin. Chem. Biol. 6:102-105; Mei et al., 2007, Biotechnol. Prog. 23:1305-1311).
In vivo protein expression systems also lack robustness and predictability due to their lack of modularity and adaptability to production at the point of need. For at least these reasons, production of proteins for such applications will not be cost effective until there are techniques and systems available to enable production of single doses or small scale made-to-order products for individual needs that meet regulatory criteria for human use.
Cell-free protein synthesis (CFPS) systems have emerged as a powerful cost-effective technology platform for rapid and efficient production of pharmaceutical proteins (Goerke and Swartz, 2008, Biotechnol. Bioeng. 99:351-367; Kanter et al., 2007, Blood. 109:3393-3399; Yang et al., 2005, Biotechnol. Bioeng. 89:503-511; Yin et al., 2012, MAbs. 4:217-225; Kline et al., 2014, Pharm. Res. 2014, PMID:25511917). CFPS systems have distinct advantages over in vivo methods for recombinant protein production (Carlson et al., 2012, Biotechnol. Adv. 30:1185-1194; Katzen et al., 2005, Trends Biotechnol. 23:150-156; Swartz 2006, J. Ind. Microbiol. Biotechnol. 33:476-485; Zawada et al., 2011, Biotechnol. Bioeng. 108:1570-1578). Cell-free systems are not constrained by ancillary processes required for cell viability and growth (e.g., homeostatic conditions), thereby allowing optimization of production for a single protein product, as well as optimization of protein complexes, incorporation of non-natural amino acids, high-throughput screening and synthetic biology. The absence of a cell membrane enables real-time monitoring, rapid sampling, purification, and direct manipulation of the protein synthesis process. In addition, the cell-free format avoids the process of cell-line generation, thereby allowing system testing and acceleration of the process/product development pipelines.
However, the current eukaryotic CFPS systems suffer from laborious extract preparation procedures, low and variable product yields, expensive reagents, low protein production rates, small reaction scales, and an unproven track record of expressing complex disulfide bonded or glycosylated proteins.
Recent research advances have led to the production of robust cell-free systems for protein synthesis in high yields. These advances have been achieved by increasing reaction duration via continuous supply of substrates and removal of toxic reaction byproducts by diffusional exchange across a membrane under a continuous exchange cell-free (CECF) format (Shirokov et al., 2007, Methods Mol. Biol. 375:19-55) as well as by activating metabolic networks in vitro for energy production, and improving extract preparation procedures (Kim and Swartz, 2001, Biotechnol. Bioeng. 74:309-316; Jewett and Swartz, 2004, Biotechnol. Bioeng. 86:19-26; Jewett et al., 2008, Mol. Syst. Biol. 4, DOI: 10.1038/msb; Zawada and Swartz, 2005, Biotechnol. Bioeng. 89:407-415; Schoborg et al., 2014, Biotechnol J. 9:630-640; Hodgman and Jewett, 2013, Biotechnol. Bioeng. 110:2643-2654). Cell-free systems also represent flexible manufacturing platforms that are highly amenable to automated liquid handling.
Automated systems for high-throughput protein production and purification for structural studies have been reported (e.g., Aoki et al., 2009, Protein Expr. Purif. 68:128-136; Makino et al., 2010, Methods Mol. Biol. 607:127-147). Such systems enable rapid dialysis-mode CFPS and purification using immobilized metal affinity chromatography. However, these robotic platforms are currently non-portable and too large for point-of-care applications. In addition, use of affinity tags for protein purification can lead to products having extra amino acids not present in the natural protein sequence (Arnau et al., 2006, Protein Expr. Purif. 48:1-13). Development of microfluidic array devices for continuous-exchange, long-lasting (up to 6 hours) bacterial CFPS have also been reported (Mei et al., 2007, Biotechnol. Prog. 23:1305-1311; Mei et al., 2010, Lab Chip. 10:2541-2545). In addition, polydimethylsiloxane (PDMS)-based microreactor array chips using bacterial cell-free extracts and having a disposable reaction chamber chip have been demonstrated to be useful for hosting CFPS reactions (Yamamoto et al., 2008, Anal. Sci. 24:243-246). These microfluidic systems hold promise for high-throughput protein screening and analysis, but due to their small scale and protein yields are not adequate for production and purification of proteins at pharmaceutical levels.
Other efforts use purified translation systems to construct minimal cells using a bottom-up approach. Multiple groups have demonstrated the ability to activate protein synthesis inside of an artificial liposome to more closely mimic native conditions (Murtas et al., 2007, Biochem. Biophys. Res. Commun. 363:12-17). Although such systems hold promise for large-scale protein screening and analysis, they are not adequate for manufacturing protein biopharmaceuticals at pharmaceutically-relevant levels.
Examples of related art include: U.S. Pat. No. 7,338,789 describing methods for in vitro synthesis of biological macromolecules under conditions and in a reaction composition wherein oxidative phosphorylation is activated and protein folding is improved; U.S. Pat. No. 8,357,529 describing methods for the enhanced in vitro synthesis of biological molecules; U.S. Pat. No. 6,780,607 describing methods of production of completely post-translationally modified proteins by combination of cell-free protein synthesis and cell-free co- and post-translational modification in reticulocytes lysates, as well as methods of supplementing those lysates with endoplasmic reticulum, Golgi and plasma membranes obtained from a Chinese Hamster Ovary (CHO) cells; and U.S. Pat. No. 8,034,581 disclosing a method for insect cell-free translation and post-translation glycosylation of target proteins, as well as conditions for cell rupture and preparation of lysates carrying translation factors and factors with glycosylation activity.